邻苯二甲酸二(2-乙基己)酯对大鼠睾丸和卵巢组织激素相关蛋白表达的影响

目的:探讨邻苯二甲酸二(2-乙基己)酯(DEHP)对大鼠睾丸和卵巢组织激素相关蛋白表达水平的影响。方法:将80只5周龄SD大鼠,雌雄各半,随机分为对照组(玉米油)、DEHP低剂量组(100 mg/kg)、DEHP中剂量组(500 mg/kg)、DEHP高剂量组(1 500 mg/kg),每组雌雄各10只,每周染毒5 d,每天灌胃染毒1次,连续6周。末次染毒24 h后处死动物,提取睾丸或卵巢组织蛋白,应用Western blot分别检测睾丸组织中3β-羟基类固醇脱氢酶(3β-HSD)、促性腺激素释放激素受体(GnRHR)、卵泡刺激素受体(FSHR)及卵巢组织中黄体生成素受体(LHR)和GnRHR的蛋白表达水平。结果:与对照组比较,雄鼠睾丸组织在DEHP 500和1 500 mg/kg剂量时3β-HSD蛋白表达水平均显著升高(P均<0.01);GnRHR蛋白表达水平在1 500 mg/kg剂量组显著下降(P均<0.01);FSHR蛋白表达水平在500和1 500 mg/kg组显著下降(P<0.05或P<0.01)。雌鼠卵巢组织在DEHP 100 mg/kg剂量组时LHR和GnRHR蛋白表达水平与对照组比较差异均无统计学意义(P>0.05),在DEHP 500和1 500 mg/kg剂量组LHR表达水平比对照组下降25%~35%,GnRHR下降60%~80%(P<0.05或P<0.01)。结论:DEHP染毒可引起大鼠睾丸和卵巢组织激素相关蛋白表达水平的改变,干扰大鼠体内性激素的代谢,推测此作用与DEHP的生殖毒性存在密切关系。     

邻苯二甲酸二(2-乙基己)酯对大鼠卵巢组织凋亡基因和癌基因表达水平的影响

目的： 探讨邻苯二甲酸二(2-乙基己)酯(DEHP)对大鼠卵巢组织凋亡基因和癌基因表达水平的影响,为评价DEHP的雌性生殖毒性提供科学依据。方法：用不同剂量DEHP灌胃染毒雌性SD大鼠6周,设对照组(玉米油)、低剂量组(100 mg/kg)、中剂量组(500 mg/kg)、高剂量组(1 500 mg/kg),利用荧光定量PCR技术检测卵巢组织凋亡基因(Bcl-2、Caspase-3、Caspase-8、Caspase-9)和癌基因(c-fos、k-ras)mRNA表达的变化。结果：Bcl-2 mRNA表达水平在DEHP高剂量组显著降低,与对照组比较差异具有统计学意义(P<0.01);Caspase-3、Caspase-8和Caspase-9 mRNA表达水平随DEHP染毒剂量升高呈上升趋势,与对照组比较显著升高,差异均有统计学意义(P<0.05或P<0.01)。c-fos mRNA表达量与对照组间的差异无统计学意义(P>0.05),k-ras mRNA表达量在DEHP中剂量组和高剂量组显著高于对照组(P<0.01)。结论：DEHP可能通过线粒体途径激活Caspase通路诱导卵巢细胞凋亡,进而损害大鼠卵巢功能和生殖内分泌功能。且DEHP可激活原癌基因异常表达,具有一定的潜在致癌风险。     

长期暴露的邻苯二甲酸二 (2-乙基己基)酯在雌性大鼠体内的代谢及对肝脏的影响

目的：研究邻苯二甲酸二（2-乙基己基）酯（DEHP）长期暴露后,雌性大鼠体内的代谢情况及对肝脏的影响。方法：将Wistar雌性大鼠64只按体质量随机分为4组,分别为对照（玉米油）组和150、300、600 mg/mL DEHP染毒组,每组16只。采用灌胃方式进行染毒,每天灌胃1次,每次按2.5 μL/g给予,连续灌胃6个月。期间观察大鼠一般生长状况,分别在染毒后3个月和6个月两个时间点采集大鼠尿液。6个月后处死各组大鼠,摘取大鼠的肝、脾和肾称取质量,计算脏体比值;并制备肝组织切片,采用HE染色法观察肝组织形态变化。对采集的2个时间点大鼠尿液,采用高效液相色谱的方法检测尿中DEHP及其代谢物邻苯二甲酸单（乙基己基）酯（MEHP）的含量。结果：与对照组相比,各剂量染毒组大鼠一般生长情况无明显差异,但肝脏的脏体比值增加,HE染色显示300和600 mg/mL DEHP染毒组大鼠肝脏组织轻微病理改变。与对照组相比,3个月时,300和600 mg/mL DEHP组随着染毒剂量的增加,大鼠尿液中DEHP和MEHP含量均明显增加,差异均有统计学意义（P均 < 0.05）。6个月时,600 mg/mL DEHP染毒组大鼠尿液中DEHP和MEHP含量高于其他3组,差异有统计学意义（P < 0.05）。结论：雌性大鼠长期暴露DEHP,体内DEHP和MEHP的残留和蓄积会增加,超过正常的代谢能力,可能会对肝脏造成一定程度的损伤。     

邻苯二甲酸二(2-乙基己)酯对大鼠卵巢病理和超微结构的影响

目的： 探讨邻苯二甲酸二(2-乙基己)酯(DEHP)对大鼠卵巢病理和超微结构的影响,评价DEHP对雌性大鼠的生殖毒性作用。方法：SPF级5周龄雌性SD大鼠40只,随机分为对照组(玉米油)、DEHP低剂量组(100 mg/kg)、DEHP中剂量组(500 mg/kg)和DEHP高剂量组(1 500 mg/kg),每组10只,每天灌胃1次,每周5次,连续染毒6周。染毒结束后称量大鼠体质量及卵巢质量,计算卵巢脏器系数;光镜下观察大鼠卵巢病理改变;电镜观察卵巢颗粒细胞超微结构的变化。结果：各染毒组大鼠体质量与对照组比较差异均无统计学意义(P均>0.05),高剂量组卵巢质量和脏器系数较对照组明显增加(P<0.01);普通光学显微镜检查发现DEHP处理后卵巢组织卵泡结构受损,电镜观察发现DEHP处理后颗粒细胞线粒体肿胀、线粒体嵴减少、胞浆空泡化、核染色质完全凝聚、出现凋亡小体、细胞变性坏死。结论：DEHP对大鼠卵巢毒性明显,影响卵泡发育,引起颗粒细胞凋亡。     

邻苯二甲酸(2-乙基己基)酯致畸致突变实验研究

目的:本文报道了邻苯二甲酸(2-乙基己基)酯(DEHP)小鼠致畸试验、小鼠骨髓嗜多染红细胞微核试验、小鼠骨髓细胞染色体畸变试验和Ames试验的研究结果.结果与结论:DEHP大剂量(1 000 mg/kg、2 000 mg/kg)时对小鼠有明显的胚胎毒性和致畸性.DEHP对Ames试验的4个标准菌株无明显诱变作用,但可致小鼠骨髓嗜多染红细胞微核形成,并具染色体畸变效应.     

邻苯二甲酸单(2-乙基)己酯对MCF-7细胞DNA的氧化损伤

目的:研究邻苯二甲酸单(2-乙基)己酯(mono-2-ethylexyl phthalate,MEHP)对人乳腺癌MCF-7细胞DNA氧化损伤的作用。方法:分别用不同浓度的MEHP(6.25、12.5、25、50和100μmol/L)和二甲基亚砜(溶剂对照,0.1%)处理MCF-7细胞12和24 h后用MTT比色法检测细胞的存活率。再分别于染毒后1.5、3、6、12和24 h,测定丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)和细胞8-羟基脱氧鸟苷(8-hydroxy-2′-deoxyguanosine,8-OHdG)水平。结果:与对照组比较,MEHP染毒12 h,6.25和12.5μmol/L组细胞的存活率显著增加(P0.05)。MEHP染毒24 h,在较高浓度处理组(≥25μmol/L)的细胞存活率显著降低(P0.05);各处理组细胞的SOD活性有所增加,细胞MDA水平、GSH-Px活性以及8-OHdG生成量无显著变化(P0.05)。当MEHP染毒MCF-7细胞较短时间(1.5、3和6 h)时,各处理组MDA水平和8-OHdG生成量均显著升高(P0.05),而SOD和GSH-Px活性则显著性降低(P0.05)。结论:一定浓度MEHP作用在短时间内(1.5、3和6 h)可引起MCF-7细胞的氧化应激反应,并触发细胞DNA的氧化性损伤,具体调控机制有待深入研究。     

邻苯二甲酸二(2-乙基己)酯对小鼠肺细胞DNA的损伤作用

目的： 探讨邻苯二甲酸二(2-乙基己)酯(DEHP)对小鼠肺细胞DNA的损伤作用。方法：以生理盐水作为阴性对照组,用不同浓度DEHP(5、25、125、625 μmol/L)对小鼠肺细胞进行体外染毒,应用单细胞凝胶电泳技术检测肺细胞DNA的断裂程度。结果：当DEHP的浓度为125和625 μmol/L时,肺细胞尾部DNA百分含量、尾矩和Olive 尾矩与阴性对照组相比均升高,差异具有统计学意义(P＜0.05)。结论：DEHP在体外可造成小鼠肺细胞的DNA断裂损伤。     

邻苯二甲酸二(2-乙基)己酯长期暴露对成年雄鼠甲状腺激素水平的影响

目的：探讨邻苯二甲酸二（2-乙基）己酯（DEHP）长期暴露对成年雄性大鼠甲状腺激素水平产生的影响。方法：将36只健康的成年雄性Wistar大鼠按体质量随机分为4组，分别为对照组以及150、300、600 mg/kg DEHP暴露组。对照组大鼠正常饮食和饮水，暴露组大鼠连续灌胃给予DEHP 3个月。处死大鼠前称取大鼠体质量并计算各组织脏体比，检测尿碘含量，血清甲状腺激素含量及血清TTR、TRH、TSAb水平。采用单因素方差分析法分析各指标的组间差异，进一步的多重比较采用SNK检验。结果：经过3个月的DEHP暴露，大鼠体质量无明显变化。与对照组相比，各剂量组大鼠甲状腺湿重和脏体比无显著性差异。150、300、600 mg/kg剂量组大鼠肝脏脏体比增加并呈剂量-效应关系（P < 0.05）。600 mg/kg剂量组大鼠尿碘水平显著降低（P < 0.05）。600 mg/kg剂量组血清FT4（血清游离甲状腺素）水平降低（P < 0.05）。结论：DEHP 3个月的暴露期对大鼠体质量未产生明显影响，对甲状腺、肝脏脏器系统功能产生一定的影响。DEHP的暴露可抑制大鼠体内TSH的合成、分泌及转运，扰乱甲状腺激素水平。     

邻苯二甲酸二(2-乙基己)酯对小鼠睾丸损伤及 p-CREB蛋白 表达的影响

目的：探讨邻苯二甲酸二（2-乙基己）酯（DEHP）对小鼠睾丸损伤及睾丸组织磷酸化环磷酸腺苷反应元件结合蛋白（p-CREB）表达的影响。方法：选用ICR初断乳雄性小鼠40只,随机分为对照组（玉米油）和DEHP染毒组（250、500、1 000 mg/kg）,经口灌胃,1次/天,6天/周,连续8周,于末次染毒24 h后处死动物,称取睾丸、附睾质量计算脏器系数并进行精子计数,检测睾丸组织形态学改变,采用免疫组织化学SP法检测睾丸组织p-CREB蛋白的表达。结果：与对照组相比,250、500和1 000 mg/kg DEHP染毒组小鼠附睾脏器系数及精子计数均明显降低（P < 0.05）,1 000 mg/kg组小鼠睾丸系数亦明显下降,差异有统计学意义（P < 0.05）。各DEHP染毒组小鼠睾丸曲细精管上皮细胞出现层次减少、排列疏松、紊乱等改变;随着染毒剂量升高,睾丸组织中p-CREB蛋白表达下降,DEHP 500和1 000 mg/kg组较对照组差异有统计学意义（P < 0.05）。结论：DEHP可致小鼠曲细精管生精上皮细胞损伤、精子数下降、p-CREB蛋白表达减少。     

邻苯二甲酸二(2-乙基己基)酯对秀丽线虫生殖能力的影响

目的：探讨邻苯二甲酸二（2-乙基己基）酯（DEHP）对秀丽线虫生殖能力的影响。方法：设置对照组（K溶液）和DEHP低（0.1 mg/L）、中（1.0 mg/L）、高（10.0 mg/L）3个剂量的染毒组,20℃下恒温培养染毒48 h,观察DEHP对秀丽线虫后代数目、世代时间以及瞬时产卵量的影响;利用二脒基苯基吲哚（DAPI）染色,观察并计数线虫有丝分裂区、减数分裂区的生殖细胞数;利用MD701突变株,在荧光显微镜下观察粗线期细胞的凋亡情况。结果：与对照组相比,L4期线虫暴露DEHP 48 h后,秀丽线虫的后代数目在1.0和10.0 mg/L剂量组显著下降（P < 0.05）,但对世代时间无明显影响（P>0.05）。随着DEHP暴露剂量的增加,线虫的瞬时产卵量下降（P < 0.05）。与对照组相比,在DEHP 1.0和10.0 mg/L的暴露剂量下,秀丽线虫总生殖细胞数和有丝分裂区生殖细胞数均显著下降（P < 0.01）,在10.0 mg/L暴露剂量下减数分裂区生殖细胞显著减少（P < 0.01）。随着染毒剂量的升高,粗线期生殖细胞凋亡数明显增加（P < 0.01）。结论：DEHP可以导致秀丽线虫有丝分裂区增殖阻滞以及减数分裂区生殖细胞数减少,这种作用可能和DEHP导致秀丽线虫粗线期凋亡数目增多有关。     

DEHP及MEHP对小鼠卵巢颗粒细胞分泌功能的影响

目的：研究邻苯二甲酸二（2_乙基）己酯（DEHP）及其活性中间产物邻苯二甲酸单（2_乙基）己酯（MEHP）对卵巢颗粒细胞及其激素合成、分泌功能的影响。方法：体外培养健康初断乳ICR小鼠的卵巢颗粒细胞,分别加入DEHP（10、50、250nmol/L）,MEHP（10、50、250nmol/L）和溶剂对照（DMSO）,作用细胞24h。用RT-PCR法测定原代小鼠颗粒细胞中类固醇激素合成急性调节蛋白（StAR）和P450侧链裂解酶（P450scc）mRNA水平;用Realtime-PCR法测定原代小鼠颗粒细胞中芳香化酶（CYP19）和过氧化物酶体增殖剂激活受体（PPARα、PPARβ、PPARγ）mRNA水平;用酶联免疫法（EIA）检测卵巢颗粒细胞上清液中雌二醇和孕酮的水平。结果：与对照组比较,250nmol/LMEHP抑制StAR基因及P450scc基因表达（P均〈0.05）;250nmol/LDEHP对CYP19基因表达有促进作用（P〈0.05）;而10、50nmol/LDEHP及50、250nmol/LMEHP对CYP19基因表达均有抑制作用（P〈0.05）。除10nmol/LDEHP外,DEHP及MEHP其它浓度组均能抑制PPARα、PPARβ基因的表达（P〈0.05）;10nmol/LDEHP及MEHP对PPARγ基因表达有促进作用（P〈0.05）,而250nmol/LMEHP对PPARγ基因表达有抑制作用（P〈0.05）。各剂量DEHP及MEHP均能使颗粒细胞分泌雌二醇增加（P〈0.05）,10nmol/LDEHP、50及250nmol/LMEHP抑制颗粒细胞分泌孕酮（P〈0.05）。结论：DEHP及MEHP影响颗粒细胞类固醇激素合成酶、PPARs的基因表达及分泌功能。     

Promoting Effect of the Peroxisome Proliferator, Clofibrate, but Not Di(2-ethylhexyl)phthalate, on Urinary Bladder Carcinogenesis in F344 Rats Initiated by N-Butyl-N-(4-hydroxybutyl)nitrosamine

The modifying potential of clofibrate and di(2-ethylhexyl)phthalate (DEHP) on second stage, N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-initiated urinary bladder carcinogenesis was investigated in male F344 rats, using a uracil-accelerated transitional cell proliferation model. Six-week-old animals received 0.05% BBN in their drinking water for 4 weeks and then clofibrate (1.0, 0.5, and 0.25%) and DEHP (1.2, 0.6, and 0.3%) were given during experimental weeks 5–8 and weeks 12–20. Uracil was administered during weeks 9–11 at a dietary level of 3.0%. Control rats were treated with BBN and uracil without peroxisome proliferator. Surviving animals were killed at the end of week 20 of the experiment, when the densities of putative preneoplastic, papillary or nodular (PN) hyperplasias (numbers per 10 cm of basement membrane) were significantly increased in all clofibrate-treated, but not the DEHP groups. The incidences of PN hyperplasia were similar in both treated animals and controls. In a second experiment, rats fed diets containing 1.0% clofibrate or 1.2% DEHP were assessed for levels of DNA synthesis in urinary bladder epithelium by 5-bromo-2'-deoxyuridine immunohistochemistry. Numbers of labeled nuclei remained within normal levels, and no proliferative changes were evident. Thus, the present experiments indicated that while clofibrate, but not DEHP, exerts weak enhancing effects on BBN-initiated urinary bladder carcinogenesis in rats this is not associated with increased levels of DNA synthesis in the affected epithelium.     

The Promoting Effects of Food Dyes, Erythrosine (Red 3) and Rose Bengal B (Red 105), on Thyroid Tumors in Partially Thyroidectomized N-Bis(2-hydroxypropyl)- nitrosamine-treated Rats

The effects of erythrosine (Red 3), rose bengal B (Red 105) and thyroidectomy on the development of thyroid tumor were examined in male Wistar rats treated with N-bis(2-hydroxypropyl)nitrosamine (DHPN). Red 3 and Red 105 were used at 4% in the basal diet and were administered for 19 weeks from week 2 to 20. Thyroidectomy was performed by resection of the left lobe at week 4. Single injection of DHPN was performed intraperitoneally at 280 mg per 100 g body weight at the beginning of the experiment. Red 3 and Red 105 significantly promoted the development of thyroid tumors in thyroidectomized rats given DHPN, but had no significant effect in non-thyroidectomized rats. The incidence of thyroid tumors was 91% in rats with partial thyroidectomy, Red 3 and DHPN, 100% in rats with partial thyroidectomy, Red 105 and DHPN, and 64% in rats with partial thyroidectomy and DHPN. Serum TSH was 5.5±3.1 ng/ml in rats with partial thyroidectomy, Red 3 and DHPN, 2.1 ± 2.2 ng/ml in rats with partial thyroidectomy, Red 105 and DHPN, and 1.5±0.5 ng/ml in rats with partial thyroidectomy and DHPN.     

米非司酮、维生素D3对乳腺癌细胞株MCF-7生长和凋亡的影响

目的:探讨米非司酮(RU486)和维生素D3(VitD3)对乳腺癌细胞株MCF-7生长和凋亡的影响,尤其是二者高浓度联合应用与单独高浓度应用之间的差异。方法:将MCF-7细胞株分别加入乙醇、VitD3和RU486。细胞培养24小时、48小时和72小时置光镜下观察细胞形态,利用体视学的方法进行计算细胞生存抑制率。72小时送流式细胞仪检查细胞分期、细胞凋亡率,经免疫组化、PI染色后行p53蛋白水平测定。结果:24小时、48小时、72小时低浓度组细胞生存抑制率与对照组乙醇相比较变化不大,而高浓度组和对照组相比较细胞生长抑制率明显升高。48小时比24小时抑制作用明显,72小时亦比48小时抑制作用明显。VitD310-7M RU48610-5M组比VitD310-7M组和RU48610-5M组72小时后的细胞生长抑制率明显升高。VitD310-7M组、RU48610-5M组、VitD310-7M RU48610-5M组较乙醇对照组相比,p53蛋白活化分子含量明显降低。结论:(1)RU486及VitD3的抗肿瘤作用有时间依赖性和剂量依赖性。(2)经析因分析后发现VitD310-7M和RU48610-5M两组具有交互作用(P<0.05),联合应用二者可以提高抗肿瘤作用。(3)RU486和VitD3可以引起乳腺癌细胞的凋亡,可能是通过一种或几种途径最终引起了p53蛋白的减少,推测其基因属突变型。     

Long-term effects of the mammary carcinogen 7,12-dimethylbenz(a) anthracene on hypothalamic gonadotropin-releasing hormone and its pituitary receptor gene expression,during the promotion stage,in female Sprague-Dawley rats

A single intragastric administration of 7,12-dimethylbenz(a)anthracene (DMBA) has been shown to induce mammary tumors in young cycling female Sprague-Dawley rats. The appearance of these tumors is preceded by a series of neuroendocrine disturbances, including attenuation of the preovulatory luteinizing hormone (LH) surge and amplification of the preovulatory 17-estradiol surge, and gonadotropin-releasing hormone (GnRH) released in vitro. In this study, we examined the hypothesis that DMBA administration decreases levels of GnRH mRNA in the preoptic area-anterior hypothalamus (POA-AH) and GnRH receptor (GnRH Rc) mRNA and protein in the anterior pituitary gland. Sprague-Dawley rats, 55–60 days of age with regular estrous cycles, received a single dose of 15mg DMBA in 1ml sesame oil delivered by intragastric intubation. A first series of experiments was performed for the measurement of hypothalamic GnRH mRNA and pituitary GnRH Rc mRNA levels. A second series of experiments was performed for the measurement of pituitary GnRH receptor. In both experiments, animals were sacrificed by decapitation at 11.00, 16.00, 18.00 and 20.00h on each day of the 7th or 8th estrous cycle (28–32 days) after treatment. GnRH and GnRH receptor mRNAs were quantified using solution hybridization-RNase protection assay. The GnRH Rc was quantified using the 125I-D-Ala⁶-N-Met-Leu⁶-des-Gly¹⁰-ethylamide GnRH. DMBA-treatment produced no significant effect on the overall mean values of GnRH mRNA. GnRH mRNA levels in control rats rose significantly between 16.00 and 20.00h on proestrus and between 18.00 and 20.00h on diestrus I. DMBA-treated rats had a surge in GnRH mRNA levels at 18.00h on proestrus, and showed additional surges at 18.00h on diestrus II and estrus. GnRH receptor mRNA content in the anterior pituitary gland surged at 16.00h on certain days of the cycle in both groups of rats. In control rats, only the surge on diestrus II proved significant, whereas DMBA-treated rats exhibited significant surges on diestrus I, diestrus II and proestrus. GnRH receptor mRNA values were significantly lower on both days of diestrus in DMBA-treated rats compared with controls. GnRH Rc peptide content, like GnRH receptor in RNA surged at 16.00h in both groups with the exception of a marked fall on proestrus day for DMBA treated rats. A reduction in the amplitude of the surge was also seen on the day of estrous and to a lesser extend on the day of diestrus DII in DMBA treated animal. Overall, there was a disruption of the GnRH Rc pattern which culminate on the day of proestrus in DMBA-treated animals. Interestingly, the daily rise between 11.00 and 16.00h which is the more pronounced on the day of proestrus in control animals, was completely blunted in DMBA-treated rats. Overall, the results are consistent with the hypothesis that the carcinogen attenuates, directly or indirectly, preovulatory biosynthesis of the GnRH receptor and LH release.